A new focal adhesion protein that interacts with integrin-linked kinase and regulates cell adhesion and spreading.
Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. ... CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH(2)-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti-CH-ILKBP antibodies, and green fluorescent protein-CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP-ILK-PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.
Mesh Terms:
Actinin, Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Calcium-Binding Proteins, Carrier Proteins, Cell Adhesion, Cell Compartmentation, Cell Size, Cricetinae, Cytoskeleton, DNA-Binding Proteins, Focal Adhesions, Humans, Microfilament Proteins, Molecular Sequence Data, Protein Binding, Protein-Serine-Threonine Kinases, Sequence Homology, Amino Acid, Two-Hybrid System Techniques
Actinin, Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Calcium-Binding Proteins, Carrier Proteins, Cell Adhesion, Cell Compartmentation, Cell Size, Cricetinae, Cytoskeleton, DNA-Binding Proteins, Focal Adhesions, Humans, Microfilament Proteins, Molecular Sequence Data, Protein Binding, Protein-Serine-Threonine Kinases, Sequence Homology, Amino Acid, Two-Hybrid System Techniques
J. Cell Biol.
Date: Apr. 30, 2001
PubMed ID: 11331308
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